Borrelia burgdorferi, the Lyme disease spirochete, is maintained in nature via a complex enzootic cycle that typically involves wild rodents and Ixodes ticks. To sustain itself, the spirochete must not only adapt physiologically to two markedly different host milieus, but it must also express virulence determinants and evade immune-mediated clearance mechanisms during mammalian infection. A finely orchestrated expression of arthropod- and mammalian host-specific genes, a process termed 'differential gene expression', is now believed to be responsible for many of these physiological, ultrastructural, and virulence-associated adaptations. Over the past several years, we and a number of other researchers have generated a substantial body of evidence that the bacterium's complement of linear and supercoiled plasmids comprises the primary substrate for genes expressed preferentially in vivo; this notion, however, requires more stringent examination. Equally important, despite the availability of the complete genomic sequence for the B31-MI isolate, Lyme disease researchers have made only modest progress in delineating and functionally characterizing the spirochete's complex array of differentially expressed genes, and we know even less about the mechanisms which regulate and coordinate these genetic programs. The underlying hypothesis of our proposal is that plasmid-encoded, differentially expressed genes are essential to the Lyme disease spirochete's parasitic strategy within the mammalian host. To address this hypothesis and its corollaries, we will characterize the differentially expressed portion of the B. burgdorferi proteome using two-dimensional electrophoresis and peptide mass fingerprinting (Specific Aim One); identify differentially expressed B. burgdorferi genes using high-density DNA microarrays (Specific Aim Two); continue our molecular and evolutionary analysis of B. burgdorferi cp32 plasmids (Specific Aim Three); and use newly developed plasmid shuttle vectors to study differential gene expression by B.burgdorferi (Specific Aim Four).